This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Neurotransmitter release at the synapse requires membrane fusion. The SNARE complex, comprised of the plasma membrane t-SNAREs Syntaxin 1A and SNAP-25 and the vesicle v-SNARE synaptobrevin, mediates the fusion of two membranes. Synaptic vesicles contain unusually high cholesterol, but the exact role of cholesterol in fusion is not known. In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposome by a factor of five at a physiological concentration. Surprisingly, however, the stimulatory effect was much more pronounced when cholesterol was on the v-SNARE side than when it was on the t-SNARE side. Site-directed spin labeling and pulsed EPR revealed that cholesterol induces a substantial conformational change of the v-SNARE transmembrane domain (TMD) from an open scissor-like dimer to a parallel dimer. When the TMD is forced to form a parallel dimer by the disulfide bond, the rate was stimulated 2.3-fold even without cholesterol, supporting the relevance of the open-to-closed conformational change to the fusion activity. The open scissor-like conformation may be unfavorable for fusion and cholesterol may relieve this inhibitory factor. The open-scissors-like conformation may force the bilayer to have negative curvature, unfavorable for fusion. But, in the presence of cholesterol, the parallel conformation of the dimer may not harbor the unfavorable negative curvature.